1. Field of the Invention
The present application concerns biology and biological chemistry and more particularly measurement of antioxidant properties of foods and biological specimens.
2. Description of Related Art
Recently there has been a revolution in human nutrition and our understanding of the relationship between human health and diet. Most of us take for granted that foods contain vitamins and that these compounds are responsible for many of the beneficial properties of food. However, this belief is a recent one. The first vitamin to be scientifically identified and described was vitamin A (retinol) which was not described until 1913. The most recently identified of the “core” vitamins is vitamin B12 which was not described until 1948. Thus, all of the major vitamins were not described until the first half of this century although the properties of certain vitamins were known considerably earlier. The knowledge that fresh fruits (particularly citrus fruits) contain some factor that prevents scurvy goes back at least several hundred years. Apocryphally, the practice of the British Navy of providing limes to their sailors on long ship voyages to prevent scurvy is responsible for the common nickname of “limeys” given to British sailor.
It seems likely that all of the “vitamins” (meaning important food factors beyond fats, carbohydrates and proteins) have not yet been discovered. This is probably at least partially responsible for the current interest in “herbal medicine” and functional foods wherein ingestion of various natural products are supposed to have particular health benefits. The studies on heart disease over the last thirty years also make it clear that not only is it important to add vitamins and other factors to the diet, it is also important to avoid certain food substances that were formerly supposed to be benign. In particular the ingestion of saturated fats, generally of animal origin, has been shown to result in artery damage and an overall lessening of cardiac fitness.
It was with some surprise that the anti-fat crusaders discovered that certain Southern European diets that are exceptionally high in saturated fats do not produce the same degree of heart disease as do fatty diets in the United States. This finding led to a search for a “protective factor” to neutralize the baneful effects of dietary fats. Several candidates rapidly came to the forefront. The Mediterranean diets are not only high in saturated fats from meats and cheeses, they are also high in mono-unsaturated fats, particularly from olive oil. There are some studies that suggest that mono or poly-unsaturated fats can at least partially neutralize the harmful effects of saturated fats. At the same time the European diet also includes a significant amount of alcohol usually in the form of red wine. There is some evidence that moderate alcohol consumption has an ameliorating influence on the circulatory system.
More importantly, perhaps, tannins or polyphenols found in red wine are powerful antioxidants. There is growing evidence that dietary antioxidants prevent a number of maladies including heart disease. Antioxidant vitamins such as vitamin C and vitamin E are strongly implicated in the prevention of heart disease and a number of other diseases. Certainly, the dietary suggestion of at least five servings per day of fruits and vegetable provides abundant antioxidants in the form of antioxidant vitamins as well as antioxidant polyphenolic compounds.
Studies of human nutrition have shown that a shortage of dietary antioxidants results in “oxidative stress” in which uncontrolled free radical production results in oxidative damage to proteins, lipids and nucleic acids. Antioxidants provide a biochemical environment that does not favor production of free radicals. Further, any free radicals that are formed are rapidly neutralized by antioxidants. Therefore, it is not surprising that a number of techniques have been developed to measure the presence of antioxidants. It is desirable to measure the antioxidant capacity of various foods as a way of estimating potential benefit from various foods or food additives. It is also beneficial to measure antioxidant levels of blood, urine, and other medical samples to asses the antioxidant status of a given patient and to determine the effect of ingesting various antioxidants on that antioxidant status.
A number of different analytic tests are used to determine antioxidant and/or “free radical trapping” content of foods. To the extent that many important antioxidants are polyphenolic compounds test, such as bromine reduction tests, for polyphenols are employed. A series of other tests are used to determine free radical trapping. The total (peroxyl) radical-trapping antioxidant potential (TRAP) assay employs 2′-azobis-(2′amidinopropane)-dihydrochloride to initiate formation of peroxyl radicals in a test substance. An oxygen electrode is then used to measure the rate that a given lipid sample resists peroxidation (e.g., by measuring the rate of oxygen uptake) in the presence of various test antioxidants. This method is generally most effective at measuring lipid soluble antioxidants and does not give a straightforward antioxidant value.
The trolox-equivalent antioxidant capacity (TEAC) is another method that measures the ability of antioxidants to prevent or quench free radicals. 2,2′azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) is reacted with hydrogen peroxide in the presence of a peroxidase enzyme to form radical cations whose presence can be detected optically by their effect on 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox). The method measures the inhibition of free radicals by antioxidants in the sample. This method requires several fairly complex reagents.
The oxygen radical absorbance capacity (ORAC) assay uses AAPH to generate peroxyl radicals. The radicals are optically measured as the fluorescence quenching and/or destruction of the algal pigment β-phycoerythrin. The assay can be automated allowing the free-radical quenching power of samples to be analyzed. Generally, instruments capable of fairly sophisticated optical measurements (e.g., fluorescence life time or total fluoresecence measurements) are required.
The total oxyradical scavaging species (TOSC) assay quantifies the reactive oxygen species scavaging potential of antioxidants. In this test ABAP is thermally decomposed to generate peroxy radicals which in turn generate ethylene gas by oxidatively decomposing α-keto-γ-methiobutyric acid. Antioxidants that scavenge the reactive oxygen prevent or diminish the formation of ethylene which is measured by a gas chromatograph. Again fairly complex and sophisticated instrumentation is required. Other complex analytic equipment such as electron spin resonance (ESR) spectrometers can also be employed to measure scavenging of free radicals by antioxidants.
Antioxidants can be measured in plasma using the fairly simple FRAP oxidation-reduction assay which optically measures the reduction of ferric-tripyridyltriazine to a ferrous form. This test is somewhat simpler than the toxic bromine test mentioned above.